Higher organisms establish cellular organization by targeting proteins to specific membrane bounded compartments. Most targeting processes involve translocation into or across a membrane bilayer. This is achieved by address signals contained within each precursor protein's primary sequence and cellular machinery to bind the signals and facilitate transmembrane transport. Many protein transport systems use ATP or GTP motors to move unfolded proteins through transmembrane channels. However, a recently discovered system called Tat, for Twin arginine translocation, is unusual because it transports fully folded proteins across sealed membranes. Protein transport is a fundamentally important process in all cells and numerous fatal human diseases result from trafficking errors. Tat systems play specific roles in infectious human diseases because certain pathogens rely on Tat for virulence. Our long range goal is to understand the mechanism of Tat protein transport using the chloroplast Tat system (called cpTat) as an experimental model. cpTat is currently the best system for biochemical dissection of Tat mechanism. cpTat operates by a cyclical process in which two subcomplexes reversibly associate to form a transient translocase, i.e. the enzyme complex that facilitates transport. A receptor complex binds the twin arginine signal and appears to present the precursor to the protein conducting component, Tha4. Although some models invoke form-fitting channels for transport, our data suggest something quite different. We found that Tha4 undergoes a major conformational change in the translocase that is not consistent with a channel organization. Indirect calibration of the translocation pathway implies a highly dynamic and transient structure. Here we propose a biochemical approach to determine characteristics of the translocase both before and during translocation with a method that can stabilize an assembled translocase. The identity of component (s) that contact of the precursor as it goes across the membrane and presumably line the pathway will be determined with specialized precursors designed to capture such interactions. The successful accomplishment of these goals will solve a longstanding scientific puzzle, may lead to highly specific therapeutic agents, and may even allow realistic engineering and defined control of nanometer sized particle transport across biological membranes. Protein transport is a fundamentally important process in all cells and numerous fatal human diseases result from trafficking errors. Tat protein transport systems play specific roles in health because certain pathogens employ Tat for virulence. Examples include Pseudomonas aeruginosa (Voulhoux et al., 2001), Mycobacterium tuberculosis (McDonough et al., 2005), E. coli 0157:H7 (Pradel et al., 2003), Legionella pneumophila (Legionnaires disease) (De Buck et al., 2005), and Helicobacter pylori (Olson and Maier, 2002). Therapeutic agents that target the Tat system of pathogens are likely to have fewer side effects because Tat systems are absent from animals. Public Health Relevance: Our studies could lead to such agents and may also lead to strategies for engineering nano-particle transport across biological membranes.